The long-range goal of this research is to develop an enzyme replacement therapy (ERT) technology for genetic metabolic diseases that is effective in mitigating the problem of immune sensitization that has hindered previous ERT technologies. ERT drugs have been approved for treating almost a dozen lysosomal diseases (LDs), however, the induction of patient anti-ERT antibodies has emerged as a significant limitation for some of these drugs reducing their effectiveness. Because early/infantile-onset forms of LDs comprise the most severe mutations, the development of immune sensitization is more prevalent in younger patients. These children often show dramatic life-saving improvements upon treatment onset, however, progress may stop and quickly decline if these patients develop neutralizing antibodies to the ERT drug. Most current ERTs for LDs exploit the Mannose-6-Phospate (M6P) receptor for uptake into disease cells and the predominant class of anti-ERT antibodies interfere with this uptake process. However, the technology developed by BioStrategies LC uses an alternative plant RTB lectin-based mechanism for ERT cell uptake. Using the Hurler MPS I model we have shown in both MPS I lysosomal disease cell cultures and subsequent short-term in vivo mouse Phase I SBIR studies of IDUA:RTB that active enzyme is successfully delivered by RTB in the presence of neutralizing antibodies against mammalian cell derived IDUA (mcd-IDUA). Based on these promising preliminary results, our goal in this SBIR Phase II follow-on project is to demonstrate bio-distribution and long-term therapeutic effectiveness of RTB delivered ERT in Hurler mice at the high level of rigor that would support FDA approval to begin IND clinical trials. Specific aims of this Phase II project are to: 1) Assess short-term biodistribution and pharmacodynamics comparing our IDUA:RTB drug to the mcd-IDUA drug in immune-sensitized Hurler mice, and 2) Evaluate effects of anti-drug antibodies following long-term administration of the IDUA-RTB verses mcd-IDUA drugs. Success in these experiments would lay the foundation for further preclinical research leading to a successful IND application to FDA to initiate clinical trials in immune compromised Hurler patients. Our broader long-term goal is to develop this new ERT delivery system for treating immune-sensitized patients for other lysosomal and metabolic diseases.